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J. Agric. Food Chem. 2006, 54, 1814−1821
Release and Perception of Ethyl Butanoate during and after Consumption of Whey Protein Gels: Relation between Textural and Physiological Parameters MONTSERRAT MESTRES,† ROLF KIEFFER,‡
AND
ANDREA BUETTNER*,‡
Deutsche Forschungsanstalt fuer Lebensmittelchemie, Lichtenbergstr. 4, D-85748 Garching, Germany, and Facultat d’Enologia, Dept Quı´mica Analı´tica i Orga`nica, Universitat Rovira i Virgili, C/Marcel‚lı´ Domingo S/N, 43007 Tarragona, Spain
The influence of gel texture on parameters such as positioning of food material in the oral cavity during mastication, and salivation, and their influence on aroma release in vivo was studied. Retronasal perception was followed by means of time-resolved sensory evaluation, while volatile release patterns were observed by means of PTR-MS. A clear correlation was found between individual-specific consumption patterns and the respective sensory perception. Also, gel texture could be clearly correlated with respective physicochemical release patterns in vivo and to the corresponding retronasal aroma perception. KEYWORDS: Time-intensity; chewing position; saliva; tooth; PTR-MS; mastication; TA; gel-texture
INTRODUCTION
A vast number of studies have dealt up to now with aroma release phenomena in vivo and their relation to sensory perception. Real food systems were investigated as well as model systems such as gels or dairy products, applying an array of different techniques such as atmospheric pressure chemical ionization (APCI-MS), proton-transfer reaction mass spectrometry (PTR-MS), exhaled odorant measurement (EXOM), and many more (1-6). However, the findings obtained often seem not fully consistent or even contradictory. Reasons for this might be, especially for in vivo analysis of aroma release, different sample consumption protocols for panelists. For example, some studies involved highly standardized guidelines for consumption but still obtained considerable variation in volatile release profiles (3, 7, 8). Completely free eating modes led to even higher variance between panelists which could not be fully explained on a physiological basis and required extensive panelist numbers with subsequent complex statistical data treatment (9). Concerning data analysis for real-time aroma release studies in vivo, recent studies furthermore showed that precise recording of events such as sample introduction or swallowing and incorporation of these parameters into final data analysis is a crucial factor for interpretation of the obtained release curves (7, 8). Subject-specific consumption patterns were also observed in a recent study on model dairy desserts, where panelists could be, consistently with their chewing patterns, grouped according to the perceived aroma intensities (3). In other gel studies, not only the influence of texture but also of artificial saliva addition and simulated mastication was * To whom correspondence should be addressed.
[email protected]. † Universitat Rovira i Virgili. ‡ Deutsche Forschungsanstalt fuer Lebensmittelchemie.
E-mail:
followed. Techniques involved a model mouth system with online monitoring of volatile release by PTR-MS analysis, while static headspace analysis was used to determine partition coefficients of compounds depending on the gel textural properties (1, 10). When observing the dynamic release from pectin gels differing in hardness in the model mouth system, reduced release of volatiles was reported when viscosity or hardness of the gels was increased (1). Also, it was found that of all parameters studied (mastication, saliva, gel hardness), mastication rate was the parameter with the largest influence on aroma release. Increase in mastication rate increased overall aroma release. This was attributed to the increase in surface area due to sample breakdown. Compared to a nonchewing condition, the maximum aroma intensity was much higher in the chewing condition and was reached later and aroma decline was much slower. On the other hand, a decrease of aroma release during mastication was found in the presence of saliva. In this context, it is interesting to note that an opposite effect was observed when the influence of artificial saliva addition on dynamic aroma release from different gels (pectin, gelatin, starch) was studied (10). Here, high aroma liberation was observed for the comparatively rigid gelatin gel, while the other softer gels remained unaffected. Generally, the effects of gel type were much more pronounced in this study as completely different gel systems were investigated than in the pectin study. The topic of the first part of this investigation (7) on model gel systems was a detailed in vivo release analysis in real time with special focus on the impact of the chewing period, termed oral or preswallow phase, the swallowing event itself, and the subsequent release after the gel bolus has been swallowed. Therefore, detailed data analysis has been developed showing
10.1021/jf0517501 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/07/2006
Aroma Release and Retronasal Aroma Perception from Flavored Gels Table 1. Rheological Properties of Gels with Different Protein Concentrations at Two Different Deformations protein content (%)
Rmax (mN)
reversible deformation (mm)
4 6 7 8 10
39 120 149 195 281
2.70 2.95 3.10 3.23 3.50
4 6 7 8 10
84 560 834 1000 1429
0.30 2.50 3.30 3.45 3.50
reversible deformation (%)
50% compression 54 59 62 65 70 80% compression 4 31 41 44 47
that averaging of overall release profiles for different panelists and calculation of the “classical” parameters Imax, Tmax, and area under the curve (AUC) was unsatisfactory. The present study will now focus on physiological parameters in relation to gel texture, such as salivary flow and chewing patterns, and their possible relevance for the respective odorant release patterns observed in vivo. Thereby, not only factors depending on sample differences, such as texture, but also those leading to variations between panelists were studied. MATERIALS AND METHODS Chemicals. Ethyl butanoate was obtained from Aldrich (Steinheim, Germany). The odorant was freshly distilled prior to analysis. Chemical and sensory purity was checked by gas chromatography-olfactometry (GC/O) as well as gas chromatography-mass spectrometry (GC-MS). Whey protein isolate (Bipro, JE 153-9-420) was obtained from Davisco Fods International, Inc., Le Sueur, MN, and glucono-δ-lactone (GDL) from Aldrich (Steinheim, Germany). Preparation of Gels. Gels with 4%, 6%, 7%, 8%, and 10% whey protein in distilled water were prepared and flavored with ethyl butanoate exactly according to the procedure described in (2). Gelling was allowed for 15 h at room temperature in open-ended syringes with an inner diameter of 9 mm for texture analysis (TA) and of 18 mm for sensory tests. To avoid contamination or aroma losses during the gelling process, the ends of the syringes were protected with Parafilm. Ethyl butanoate content in the samples was checked by means of stable isotope dilution assays according to (11). Gels were kept at 4 °C between sessions and stored at this temperature for a maximum period of 48 h. Prior to analysis, they were pulled out of the syringe by means of the piston and freshly cut into cylinder-shaped samples of 0.65 mL for TA-testing and 2 mL for determination of chewing position, for determination of salivation and also for sensory evaluation and PTRMS analyses. Texture Analysis. TA was performed by means of a texture analyzer (Stable Microsystems, U.K.) with the following parameters: temperature 22°C; test-speed for compression 0.5 and 2 mm/s for the upstroke the higher speed giving a better impression of the elastic (solid) character. The reversible deformation during upstroke was monitored by the distance until the relaxation stress of the sample was zero again and was expressed as % of the total compression (Table 1). The maximum force (Rmax (mN)) as a function of the deformation monitored the hardness of the cylindric sample of 10 mm height compressed by 50% and 80% of its original height. To get an idea of the force the tip of the tongue exerts on a piece of gel freshly introduced to the mouth, a cylindric hard piece of gel was fixed onto the hook of the SMS/ Kieffer-Rig, placed just behind the incisors and touched by the tongue as was the case during the sensory evaluations. Panelists. Seven panelists (two male, five female, age 22-40, nonsmokers) were recruited from the Technical University of Munich. They exhibited no known illnesses at the time of examination and normal olfactory and gustatory function. In regular weekly training
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sessions, panelists were tested for their sensory performance with selected suprathreshold aroma solutions prior to participation in the experiments, while subjective aroma perception was tested with a defined set of aroma substances and an internally developed “flavour language” (12). The panelists had a normal salivary flow, tested in model chewing experiments as described in (7). Intraoral analyses were performed 2 h after breakfast and thorough cleaning of the teeth and oral cavity with a commercial toothpaste (5 min). Determination of Chewing Position. Gels with 4%, 6%, 7%, 8%, and 10% protein concentration, respectively, were singly presented to the panelists in random order for uninstructed consumption. After chewing and swallowing, panelists were asked about the main chewing positions for each gel. They had not only to tell the preferred side of chewing (if any), but also to locate the area of chewing (incisors, eyeteeth, premolars, molars). Experiments were performed three times for each panelist and gel on one day and were repeated on two following days. After this, panelists were asked to chew the softest and the hardest gel, thereby deliberately changing their preferred chewing positions to the opposite. Panelists had to rate the degree of convenience/familiarity of this changed chewing pattern on a seven-point scale from 0 (unfamiliar/inconvenient) to 3 (highly familiar/convenient). Furthermore, they evaluated the overall aroma perception of gel samples on a seven-point scale (steps of 0.5 for rating) from 0.0 (not perceivable) to 3.0 (very intense) while chewing normally and also when chewing with the changed pattern. Determination of Salivation. Weighted gel samples (3 replicates each) with 4% and 10% protein concentration with and without aromatization with ethyl butanoate, respectively, were singly presented to seven panelists in random order with a 30 min break between samples. After each sample evaluation, panelists rinsed their mouths with water. Panelists were asked to chew each sample singly for 1 min according to their normal chewing behavior and to avoid swallowing during the chewing procedure. After 1 min, panelists spat out the total amount of sample and saliva present in their oral cavities and the spit-off samples were weighed again. The degree of salivation was calculated from the weight difference compared to the sample before chewing. Sensory Evaluation and PTR-MS Analysis. Sensory analyses were performed in a sensory panel room at 21 ( 1 °C at three different sessions. The samples with 4 and 10% protein content, respectively, were taken into the oral cavity and chewed for 30 s with closed lips and without swallowing. Then, panelists were instructed to swallow the entire bolus and, after that, to continue chewing for 60 s. The different gels were presented in triplicate to the panelists. The order of the gels was randomized with a 15 min break between samples, and after each evaluation, the panelists rinsed the oral cavity with tap water. No information about the purpose of the experiment or the exact composition of the samples was given to the panelists. Panelists were not specifically trained to produce TI curves but should indicate during the whole chewing procedure the moments of intense aroma perception by raising their thumbs. End of subjective aroma perception should be indicated by raising the whole hand. In parallels to sensory evaluation, nosespace air was sampled with two glass tubes fitted into the nostrils (7). The transfer line was a heated silo steel capillary with an inner diameter of 0.5 mm. A small fraction of 15 sccm was introduced into the drift tube of the PTR-MS. The tubes were heated at 50 °C to prevent condensation along the sampling line. During the whole gel chewing sequence, as described above, the nosespace volatile concentration was measured simultaneously by using real-time PTR-MS. By resting the nostrils at the glass tubes, the tidal breath flow from the nostril was directly sampled without disturbance of the normal breathing or gel consumption pattern. PTR-MS data was always recorded together with the sensory evaluation by the panelists and their respective manual aroma indications as described above. Panelists were not allowed to look at any time at the data recording system and had no visual, acoustical, or other indication on when odor signals were detected by the MS system. The PTR-MS technique has been extensively discussed in a series of review papers (13-15). Briefly, it combines a soft, sensitive, and
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Figure 1. (a) Force the tip of a tongue exerts on a hard gel during the first sensory evaluations. (b) Reversible deformation of gels during upstroke as a function of the gel−protein concentration. Diamonds ) 50% of compression, squares ) 80%. Each determination was performed in duplicate; maximum relative deviation ±4%. efficient mode of chemical ionization (CI), adapted to the analysis of trace VOCs, with a mass filter. In this study, 15 sccm gas was continuously introduced into the drift tube (CI cell). The drift tube contained, besides the buffer gas, a controlled ion density of H3O+. VOCs that have proton affinities larger than water (proton affinity of H2O: 166.5 kcal/mol) are ionized by proton transfer from H3O+, and the protonated VOCs are mass analyzed. The ion source produces nearly exclusively H3O+ ions (
